Typical methods for introducing DNA into a cell include DNA condensing reagents such as calcium phosphate, polyethylene glycol, lipid-containing reagents, such as liposomes, multi-lamellar vesicles, as well as virus-mediated strategies. However, such methods can have certain limitations. For example, there are size constraints associated with DNA condensing reagents and virus-mediated strategies. Further, the amount of nucleic acid that can be delivered into a cell is limited in virus strategies. Not all methods facilitate insertion of the delivered nucleic acid into cellular nucleic acid, and while DNA condensing methods and lipid-containing reagents are relatively easy to prepare, the insertion of nucleic acid into viral vectors can be labor intensive. Virus-mediated strategies can be cell-type or tissue-type specific, and the use of virus-mediated strategies can create immunologic problems when used in vivo.
Transposons have become a suitable tool to address these issues. Transposons, or transposable elements, include a nucleic acid sequence flanked by upstream and downstream, with terminal domain sequences. Active transposons encode enzymes that facilitate the excision and insertion of the nucleic acid into target DNA sequences.
Transposable elements represent a substantial fraction of many eukaryotic genomes. For example, about 50% of the human genome is derived from transposable element sequences, and other genomes, for example plants, may consist of substantially higher proportions of transposable element-derived DNA. Transposable elements are typically divided into two classes, class 1 and class 2. Class 1 is represented by the retrotransposons (LINEs, SINEs, LTRs, and ERVs). Class 2 includes the “cut-and-paste” DNA transposons, which are characterized by terminal inverted repeats (TIRs) and are mobilized by an element-encoded transposase. Currently, 10 superfamilies of cut-and-paste DNA transposons are recognized in eukaryotes.
Transposon vectors are a proven and viable alternative to viral vectors for stable gene delivery (Meir et al., Chang Gung Med J 34:565-579 (2011); Li et al., J. Control Release 123:181-183 (2007); Kawakami et al., J. Pharm. Sci. 97:726-745 (2008); Nakanishi et al., Mol. Ther. 18:707-714 (2010)), and provide relative advantages from the standpoints of size and integration. Like integrated viruses, transposons deliver transgenes to target cells in vitro and in vivo where they are incorporated into the host genome. Unlike viruses they do not generate an immune response, they have a simpler genome, and are easier to handle. In addition, they can hold a significantly larger transgene insert than viruses, in some cases up to 100 kilobases (Li et al., Nucleic Acids Res; 39:e148 (2011)). These characteristics make transposons an attractive option for gene delivery.
PiggyBac vectors are one of the most active and flexible class 2 transposon systems available for the stable transfection of mammalian cells (Wilson et al., Mol. Ther. 15:139-145 (2007); Wu et al., Proc. Natl. Acad. Sci. U.S.A. 103:15008-15013 (2006)). The wild type piggyBac transposon is 2,472 base pairs in length, and is composed of two inverted minimal terminal repeats (“minTR”), two internal domain sequences (“ID”) and a transposase-encoding domain (Zhuang et al., Acta Biochim. Biophys. Sin (Shanghai) 42:426-431 (2010)). Transposase catalyses the excision of the transposon from one DNA source (i.e., a delivered plasmid) and allows its subsequent re-integration into another DNA source (i.e., the host cell genome).
In the majority of piggyBac vectors, the transposase gene is removed from the transposon and replaced by transgenes of interest; the transposase is then usually delivered to the cell, typically by a separate plasmid. The minTRs and IDs are crucial for the effective integration of the transposon into the host genome and together (known as terminal domains) consist of more than 700 base pairs each (Zhuang et al., Acta. Biochim. Biophys. Sin (Shanghai) 42:426-431 (2010)). The 5′ terminal domain also serves as a native promoter for transposase expression. As part of the transposition, the terminal domains are integrated into the host cell genome, exclusively at TTAA integration site, alongside the delivered transgene of interest (Elick et al., Genetica 98:33-41 (1996); Fraser et al., Insect Mol. Biol. 5:141-151 (1996)). Therefore, like integrated viruses, they deliver a significant amount of extra DNA to the target cell genome. Although the terminal domains are required for successful transposition, once integrated into the host cell genome, they perform no useful function. In fact, they may increase the risk of insertional mutagenesis (Meir et al., BMC Biotechnol 2011; 11:28 (2011)), due to any apparent or potential promoter or enhancer activity that the terminal domains might exert on host cell oncogenes (Cadinanos et al., Nucleic Acids Res. 35:e87 (2007); Shi et al., BMC Biotechnol. 7:5 (2007). Neither the 5′ nor the 3′ piggyBac minTRs contain known active promoters or enhancers (Handler et al., Proc. Natl. Acad. Sci. USA 95:7520-7525 (1998); Shi et al., BMC Biotechnol. 7:5 (2007)).
However, attempts to reduce the size of the terminal domains to decrease this risk have resulted in a significant loss of transposition efficiency. See, e.g., Zhuang et al., Acta Biochim. Biophys. Sin (Shanghai) 42:426-431 (2010); Li et al., Insect Mol. Biol. 14:17-30 (2005)).
There still remains a need for new methods and constructs for introducing DNA into a cell, and promote the efficient insertion of DNA of varying sizes into the genome of a target cell, without sacrificing stable integration efficiency and which also decreases insertional mutagenesis and eliminates promoter/enhancer activity that the integrative sequences may have on host cell oncogenes.